Method of vaccination

ABSTRACT

The present invention provides a method of expressing an antigenic molecule or a part thereof on the surface of an antigen-presenting cell, said method comprising introducing a molecule into the cell cytosol by photochemical internalisation, wherein said molecule, or a part thereof, is subsequently presented on the surface of said cell. Methods of vaccination comprising this method, together with compositions comprising said cells and uses involving said cells expressing antigenic molecules are also provided.

The present invention relates to a method of vaccination which involvesusing photodynamic treatment (PDT) to introduce vaccine components intocells to achieve antigen presentation, and to vaccine compositionsuseful in such a method.

The majority of molecules do not readily penetrate cell membranes.Methods for introducing molecules into the cytosol of living cells areuseful tools for manipulating and studying biological processes. Amongthe most commonly used methods today are microinjection, red blood cellghost-mediated fusion and liposome fusion, osmotic lysis of pinosomes,scrape loading, electroporation, calcium phosphate and virus-mediatedtransfection. These techniques are useful for investigating cells inculture, although in many cases they may be impractical, time consuming,inefficient or they may induce significant cell death. Thus suchtechniques are not optimal for use in biological or medical research, orin therapies, where it is required that cells should remain viableand/or functional.

It is well known that porphyrins and many other photosensitizingcompounds may induce cytotoxic effects on cells and tissues. Theseeffects are based upon the fact that upon exposure to light thephotosensitizing compound may become toxic or may release toxicsubstances such as singlet O₂ or other oxidising radicals which aredamaging to cellular material or biomolecules, including the membranesof cells and cell structures, and such cellular or membrane damage mayeventually kill the cells. These effects have been utilised in thetreatment of various abnormalities or disorders, including especiallyneoplastic diseases. The treatment is named photodynamic therapy (PDT)and involves the administration of photosensitizing(photochemotherapeutic) agents to the affected area of the body,followed by exposure to photoactivating light in order to activate thephotosensitizing agents and convert them into cytotoxic form, wherebythe affected cells are killed or their proliferative potentialdiminished. Photosensitizing agents are known which will localisepreferentially or selectively to the desired target site e.g. to atumour or other lesion.

A range of photosensitizing agents are known, including notably thepsoralens, the porphyrins, the chlorins and the phthalocyanins. Suchdrugs become toxic when exposed to light.

Photosensitizing drugs may exert their effects by a variety ofmechanisms, directly or indirectly. Thus for example, certainphotosensitisers become directly toxic when activated by light, whereasothers act to generate toxic species, e.g. oxidising agents such assinglet oxygen or other oxygen-derived free radicals, which areextremely destructive to cellular material and biomolecules such aslipids, proteins and nucleic acids.

Porphyrin photosensitisers act indirectly by generation of toxic oxygenspecies, and are regarded as particularly favourable candidates for PDT.Porphyrins are naturally occurring precursors in the synthesis of heme.In particular, heme is produced when iron (Fe³⁺) is incorporated inprotoporphyrin IX (Pp) by the action of the enzyme ferrochelatase. Pp isan extremely potent, photosensitizer, whereas heme has nophotosensitizing effect. A variety of porphyrin-based orporphyrin-related photosensitisers are known in the art and described inthe literature.

The cytotoxic effect is mediated mainly through the formation of singletoxygen. This reactive intermediate has a very short lifetime in cells(<0.04 μs). Thus, the primary cytotoxic effect of PDT is executed duringlight exposure and very close to the sites of formation of ¹O₂. ¹O₂reacts with and oxidizes proteins (histidine, tryptophan; methionine,cysteine, tyrosine), DMA (guanine), unsaturated fatty acids andcholesterol. One of the advantages of PDT is that tissues unexposed tolight may be left unaffected i.e. that a selective PDT effect may beobtained. There is extensive documentation regarding use of PDT todestroy unwanted cell populations, for example neoplastic cells. Thepatent literature describes a number of photodynamic compounds, alone orconjugated with targeting agents, e.g. immunoglobulins directed toneoplastic cell receptor determinants, making the complex more cellspecific. Certain photochemical, compounds, such as hematoporphyrinderivatives, have furthermore an inherent ability to localise inmalignant cells. Such methods and compounds, are described in theNorwegian patent No 173319, in Norwegian patent applications Nos. 900731, 176 645, 176 947, 180 742, 176 786, 301 981, 30 0499 and 89 1491.

In WO93/14142 a drug delivery system is described which comprises ananti-cancer agent and a photoactivatable agent (i.e. a photosensitizer)attached to copolymeric carriers. Upon administration this complexenters the cell interior by pinocytosis or phagocytosis and locatesinside the endosomes and lysosomes. In the lysosomes, the bond betweenthe anti-neoplastic agent and the polymer is hydrolysed and the formercan diffuse passively through the lysosome membrane into the cytosol.The utility of this method is thus limited to small molecular compoundswhich are able to diffuse across the lysosome membranes. After allowinga time lag for diffusion, a light source of appropriate wavelength andenergy is applied to activate the photo-activatable compound. Thecombined effect of the anti-cancer agent and photoactivatable agentdestroy the cell.

Such PDT methods as described above are thus directed to the destructionof cell structures leading to cell death.

WO 96/07432, on the other hand, is concerned with methods which use thephotodynamic effect as a mechanism for introducing otherwisemembrane-impermeable molecules into the Cytosol of a cell in a mannerwhich does not result in widespread cell destruction or cell death. Inthis method, the molecule is co-internalised (more particularly“endocytosed”) into an intracellular vesicle in the cell (e.g. alysosome or endosome) together with a photosensitizing agent. The cellis then exposed to photoactivating light which “activates” thephotosensitizer, which in turn causes the vesicle membrane to disrupt orrupture, releasing the vesicle contents, including the molecule, intothe cell interior i.e. the cytosol. It was found that in such a methodthe functionality or the viability of the majority of the cells was notdeleteriously affected. Thus, the utility of such a method, termed“photochemical internalisation” was proposed for transporting a varietyof different molecules, including therapeutic agents, into the cytosoli.e. into the interior of a cell.

We have now found that such a method can advantageously be used, notonly to transfer molecules in the interior of a cell, but also topresent or express them on a cell surface. Thus, following transport andrelease of a molecule into the cell cytosol, it may be transported tothe surface of the cell where it may be presented on the outside of thecell i.e. on the cell surface. Such a method has particular utility inthe field of vaccination, where vaccine components i.e. antigens orimmunogens, may be introduced to a cell for presentation on the surfaceof that cell, in order to induce, facilitate or augment an immuneresponse.

At its most general, the present invention thus provides a method ofexpressing an antigenic molecule or a part thereof on the surface of acell, preferably an antigen-presenting cell, said method comprisingintroducing a molecule into the cell cytosol by photochemicalinternalisation, wherein said molecule, or a part thereof, issubsequently presented on the surface of said cell.

As used herein “expressing” or “presenting” refers to the presence ofthe molecule or a part thereof on the surface of said cell such that atleast a portion of that molecule is exposed and accessible to theenvironment surrounding that cell. Expression on the “surface” may beachieved in which the molecule to be expressed is in contact with thecell membrane and/or components which may be present or caused to bepresent in that membrane.

Such antigenic presentation may advantageously result in the stimulationof an immune response, preferably an immune response which confersprotection against subsequent challenge by an entity comprising orcontaining said antigen molecule or part thereof, and consequently theinvention finds particular utility as a method of vaccination.

More particularly, this aspect of the invention provides a method ofexpressing an antigenic molecule or apart thereof on the surface of acell, said method comprising:

contacting said cell with said antigenic molecule and with aphotosensitizing agent, wherein said molecule and said agent ate eachtaken up into an intracellular membrane-restricted compartment of saidcell; and

irradiating said cell with light of a wavelength effective to activatethe photosensitizing agent, such that the membrane of said intracellularcompartment is disrupted, releasing said-molecule into the cytosol ofthe cell, without killing the cell,

wherein, said released antigenic molecule, or a part thereof, issubsequently presented on the surface of said cell.

As used herein, a “disrupted” compartment refers to destruction of theintegrity of the membrane of that compartment either permanently ortemporarily, sufficient to allow release of the antigenic moleculecontained within it.

Alternatively viewed, this aspect of the invention also provides acomposition for use in expressing an antigenic molecule or a partthereof on the surface of a cell, preferably to simulate an immuneresponse, said composition comprising an antigenic molecule and aphotosensitizing agent. Preferably said composition is pharmaceuticallyacceptable and contains also a pharmaceutically acceptable excipient ordiluent.

In a further aspect, the invention also provides the use of an antigenicmolecule and a photosensitizing agent in the preparation of a medicamentfor use in expressing said antigenic molecule or a part thereof on thesurface of a cell to stimulate an immune response.

A still further aspect of the invention provides a product comprising anantigenic molecule and a photosensitizing agent as a combinedpreparation for simultaneous, separate or sequential use in expressingsaid antigenic molecule or a part thereof on the surface of a cell,preferably to stimulate an immune response.

A yet further aspect of the invention provides a kit for use inexpressing an antigenic molecule or a part thereof on the surface of acell, said kit comprising

a first container containing said antigenic molecule; and

a second container containing a photosensitizing agent.

In the invention, the antigenic molecule may be any molecule whereinthat molecule or a part thereof is capable of stimulating an immuneresponse, when presented to the immune system in an appropriate manner.Advantageously, therefore the antigenic molecule will be a vaccineantigen or vaccine component, such as a polypeptide containing entity.

Many such antigens or antigenic vaccine components are known in the artand include all manner of bacterial or viral antigens or indeed antigensor antigenic components of any pathogenic species including protozoa orhigher organisms. Whilst traditionally the antigenic components ofvaccines have comprised whole organisms (whether live, dead orattenuated) i.e. whole cell vaccines, in addition sub-unit vaccines,i.e. vaccines based on particular antigenic components of organisms e.g.proteins or peptides, or even carbohydrates, have been widelyinvestigated and reported in the literature. Any such “sub-unit”-basedvaccine component may be used as the antigenic molecule of the presentinvention. However, the invention finds particular utility in the fieldof peptide vaccines. Thus, a preferred antigenic molecule according tothe invention i.e. a peptide (which is defined herein to includepeptides of both shorter and longer lengths i.e. peptides, oligopeptidesor polypeptides, and also protein molecules or fragments thereof e.g.peptides of 5-500 e.g. 10 to 250 Such as 15 to 75, or 8 to 25 aminoacids). Parts of antigenic molecules which are presented or expressedpreferably comprise parts which are generated by antigen-processing,machinery within the cell. Parts may however be generated by other meanswhich may be achieved through appropriate antigen design (e.g. pHsensitive bands) or through other cell processing means. Convenientlysuch parts are of sufficient size to generate an immune response, e.g.in the case of peptides greater than 5, e.g. greater than 10 or 20 aminoacids in size.

A vast number of peptide vaccine candidates have been proposed in theliterature, for example in the treatment of viral diseases andinfections such as AIDS/HIV infection or influenza, canine parvovirus,bovine leukaemia virus, hepatitis, etc. (see e.g. Phanuphak et al.,Asian Pac. J. Allergy. Immunol. 1997, 15(1), 41-8; Naruse, HokkaidoIgaku Zasshi 1994, 69(4), 811-20; Casal et al., J. Virol., 1995, 69(11),7274-7; Belyakov et al., Proc. Natl. Acad. Sci. USA, 1998, 95(4),1709-14; Naruse et al., Proc. Natl. Sci. USA, 1994 91(20), 9588-92;Kabeya et al., Vaccine 1996, 14(12), 1118-22; Itoh et al., Proc. Natl.Acad. Sci. USA, 1986, 83(23) 9174-8. Similarly bacterial peptides may beused, as indeed may peptide antigens derived from other organisms orspecies.

In addition to antigens derived from pathogenic organisms, peptides havealso been proposed for use as vaccines against cancer or other diseasessuch as multiple sclerosis. For example, mutant oncogene peptides holdgreat promise as cancer vaccines acting an antigens in the simulation ofcytotoxic T-lymphocytes. (Schirrmacher, Journal of Cancer Research andClinical Oncology 1995, 121, 443-451; Curtis Cancer Chemotherapy andBiological Response Modifiers, 1997, 17, 316-327). A synthetic peptidevaccine has also been evaluated for the treatment of metastatic melanoma(Rosenberg et al., Nat. Med. 1998, 4(3), 321-7). A T-cell receptorpeptide vaccine for the treatment of multiple sclerosis is described inWilson et al., J. Neuroimmunol. 1997, 76(1-2), 15-28. Any such peptidevaccine component may be used as the antigenic molecule of theinvention, as indeed may any of the peptides described or proposed aspeptide vaccines in the literature. The peptide may thus be synthetic orisolated or otherwise derived from an organism.

The cell which is subjected to the methods, uses etc. of the inventionmay be any cell which is capable of expressing, or presenting on itssurface a molecule which is administered or transported into itscytosol.

Since the primary utility of the invention resides inantigen-presentation or vaccination, the cell is conveniently an immuneeffector cell i.e. a cell involved in the immune response. However,other cells may also present antigen to the immune system and these alsofall within the scope of the invention. The cells according to thepresent invention are thus advantageously antigen-presenting cells. Theantigen-presenting cell may be involved in any aspect or “arm” of, theimmune response, including both humoral and cell-mediated immunity, forexample the stimulation of antibody production, or the stimulation ofcytotoxic or killer cells, which may recognise and destroy (or otherwiseeliminate) cells expressing “foreign” antigens on their surface. Theterm “stimulating an immune response” thus includes all types of immuneresponses and mechanisms for stimulating them.

The stimulation of cytotoxic cells or antibody-producing cells, requiresantigens to be presented to the cell to be stimulated in a particularmanner by the antigen-presenting cells, for example MHC Class Ipresentation (e.g. activation of CD8⁺ cytotoxic T-cells requires MHC-1antigen presentation).

Antigen-presenting cells are known in the art and described in theliterature and include for example, lymphocytes (both T and B cells),dendritic cells, macrophages etc. Others include for example cancercells e.g. melanoma cells.

For antigen presentation by an antigen-presenting cell to a cytotoxicT-cell (CTL) the antigenic molecule needs to enter the cytosol of theantigen-presenting cell (Germain, Cell, 1994, 76, 287-299). The presentinvention provides an efficient means of delivery of the antigenicmolecule into the cytosol.

Once released in the cell cytosol by the photochemical internalisationprocess, the antigenic molecule may be processed by theantigen-processing machinery of the cell and presented on the cellsurface in an appropriate manner e.g. by Class I MHC. This processingmay involve degradation of the antigen, e.g. degradation of a protein orpolypeptide antigen into peptides, which peptides are then complexedwith molecules of the MHC for presentation. Thus, the antigenic moleculeexpressed or presented on the surface Of the cell according to thepresent invention may be a part or fragment of the antigenic moleculewhich is internalised (endocytosed).

Antigens may be taken up by antigen-presenting cells by endocytosis anddegraded in the endocytic vesicles to peptides. These peptides may bindto MHC class II molecules in the endosomes and be transported to thecell surface where the peptide-MHC class II complex may be recognised byCD4+ T helper cells and induce an immune response. Alternatively,proteins in the cytosol may be degraded, e.g. by proteasomes andtransported into endoplasmic reticulum by means of TAP (transporterassociated with antigen presentation) where the peptides may bind to MHCclass I molecules and be transported to the cell surface as illustratedin the FIG. 1 (Yewdell and Bennink, 1992, Adv. Immunol. 52: 1-123). Ifthe peptide is of foreign antigen origin, the peptide-MHC class Icomplex will be recognised by CD8+ cytotoxic T-cells (CTLs). The CTLswill bind to the peptide-MHC (HLA) class I complex and thereby beactivated, start to proliferate and form a clone of CTLs. The targetcell and other target cells with the same peptide-MHC class I complex onthe cells surface may be killed by the CTL clone. Immunity against theforeign antigen may be established if a sufficient amount of the antigencan be introduced into the cytosol (Yewdell and Bennink, 1992, supra;Rock, 1996, Immunology Today 17: 131-137). This is the basis fordevelopment of inter alia cancer vaccines. One of the largest practicalproblems is to introduce sufficient amounts of antigens (or parts of theantigen) into the cytosol. This may be solved according to the presentinvention by PCI. This principle is illustrated in FIG. 1, which showshow PCI can be utilised to stimulate CTLs. A peptide or protein (P) isapplied extracellularly to antigen-presenting cells. P is endocytosedand released into cytosol by PCI. The peptide or protein will thereafterbe partly degraded by proteasomes and transported to the cells surfacecomplexed to MHC (HLA) class I where the complex can be recognised byCTLs.

As will be described in more detail in the Examples below, it has beendemonstrated that photochemical internalisation may be used efficientlyaccording to the present invention for cytosolic delivery ofcancer-specific peptides.

The antigenic molecule and/or photosensitivity agent may be targeted tospecific cells or tissues by employing targeting agents e.g.target-specific delivery or carrier systems or carrier molecules. Thusfor example the antigenic molecule and/or photosensitizing agent may bedelivered to the cell using a vector or carrier system e.g.reconstituted LDL-particles. The carrier molecule may be bound orconjugated to the antigenic molecule, to the photosensitizing agent orboth, and the same or different carrier molecules may be used. Theantigenic molecule and/or photosensitizing agent may also be conjugatedto a site-targeting ligand, such as a ligand which is specific forparticular cell-types or particular cell structures e.g. an antibodyrecognising a surface antigen expressed on certain cell types e.g. atumour-specific antigen. Such mechanisms may act to increase uptake ofthe photosensitizer and/or antigen molecule through receptor-mediatedendocytosis. Such targeting molecules carriers or vectors may also beused to direct the antigenic molecule and/or photosensitizing agent tothe intracellular compartment.

The intracellular membrane-restricted compartment may be any suchcompartment which is present in a cell. Preferably the compartment willbe a membrane vesicle, especially an endosome or a lysosome. However,the intracellular compartment may also include the Golgi apparatus orthe endoplasmic reticulum. All that is required is that the antigenicmolecule and the photosensitizing agent locate to the same intracellularcompartment(s).

The photochemical internalisation process is described in more detail inWO 96/07432 (the contents of which are incorporated herein byreference). Methods of PDT are also now widely described in theliterature.

The photosensitizing agent to be used according to the present inventionmay be any such agent which localises to intracellular compartments,particularly endosomes or lysosomes. A range of such photosensitizingagents are known in the art and are described in the literature,including in WO96/07432. Mention may be made this respect of di- andtetrasulfonated aluminium phthalocyanine, sulfonatedtetraphenylporphines (TPPS_(n)), nile blue, chlorin e₆ derivatives,uroporphyrin I, phylloerythrin, hematoporphyrin and methylene blue whichhave been shown to locate in endosomes and lysosomes of cells inculture. This is inmost cases due to endocytic activity.

Classes of suitable photosensitizing agent which may be mentioned thusinclude porphyrins, phthalocyanines, purpurins, chlorins,benzoporphyrins naphthalocyanines, cationic dyes, tetracyclines andlysomotropic weak bases or derivatives thereof (Berg et al.,Photochemistry and Photobiology, 1997, 65, 403-409).

Preferred photosensitizing agents include TPPS₄ (Zabner et al., J. Biol.Chem. 1995, 270, 18997-19007) TPPS_(2a) and AlPcS_(2a).

The photochemical internalisation according to the present invention maybe carried out using PDT methods which are known and standard in the artand appropriate modifications of such techniques which are effective inthis method. Thus, the antigenic molecule and photosensitizing agent maybe delivered to the cell by application or administration according tomethods and means known in the art of PDT.

The methods of the present invention may be used in vitro or in vivo,either by in situ treatment oripy ex vivo treatment, followed byadministration of the treated cells.

Thus, a further aspect of the invention provides an antigen-presentingcell expressing an antigenic molecule, or a part thereof, on itssurface, which cell is obtainable (or obtained) by a method ashereinbefore defined. Other aspects of the invention provide apopulation or culture of such cells, especially a viable andfunctionally intact population or culture of such cells, and also theuse of such a cell (or population or culture of cells) in therapy,particularly for stimulating an immune response, and especially forstimulating CTLs.

Also provided is the use of such a cell (or population or culture ofcells) for the preparation of a medicament (e.g. a vaccine composition)for stimulating an immune response, and especially for stimulating CTLs.

In vivo, any mode of administration common or standard in the art may beused, e.g. injection, infusion, topical administration, both to internaland external body surfaces etc. For in vivo use, the invention can beused in relation to any tissue which contains the target cells,including body fluid locations, as well as solid tissues. All tissuescan be treated as long as the photosensitizer is taken up by the targetcells, and the light can be properly delivered.

Thus, the compositions of the invention may be formulated in anyconvenient manner according to techniques and procedures known in thepharmaceutical art, e.g. using one or more pharmaceutically acceptablecarrier or excipients. The nature of the composition and carriers orexcipient materials, dosages etc. may be selected in routine manneraccording to choice and the desired route of administration, purpose ofvaccination etc. Dosages may likewise be determined in routine mannerand may depend upon the nature of the antigenic molecule, purpose ofvaccination, age of patient, mode of administration etc., in connectionwith the photosensitizing agent the potency/ability to disrupt membraneson irradiation, should also be taken into account.

The light irradiation step to activate the photosensitizing agent maylikewise take place according to techniques and procedures well known inthe art. For example, the wavelength and intensity of the light may beselected according to the photosensitizing agent used. Suitable lightsources are well known in the art.

As mentioned earlier, and as described in WO96/07432, it has been foundthat photochemical internalisation in this manner does not deleteriouslyaffect the viability and functionality of the cells. In particular, ithas been found that when a population or plurality of cells is treatedaccording to the present invention, a majority of the cells are notkilled, and survive the treatment, substantially functionally intact.

As used herein, the term “without killing the cell” is intended todefine such a situation. In other words in a population or plurality ofcells, substantially all of the cells, or a significant majority (e.g.at least 75%, more preferably at least 80, 85, 90 or 95% of the cells)are not killed.

Clearly when dealing with the light irradiation of a population or aplurality of cells it is possible that certain groups of cells orcertain areas of tissue may receive more light or in some other way besubjected to a larger PCI effect that other groups of cells or areas oftissue. Thus, the percentage values given for cell survival are notnecessarily uniform across the entire irradiated population and refer tothe percent of viable cells which remain in the irradiated population,the requirement being only that a sufficient portion of the irradiatedcells survive. In addition, cell death induced by irradiation may takesome time, e.g. a number of hours to occur. In this case it can be seenthat cells which eventually die might also be able to express anantigenic molecule on their surface in accordance with the methods ofthe present invention and may thus be involved in the methods, uses etc.of the present invention. Thus the % cell death refers to the percent ofcells which remain viable within a few hours of irradiation (e.g. up to4 hours after irradiation) but preferably refers to the % viable cells 4or more hours after irradiation.

The methods of the invention may be modified such that the fraction orproportion of the surviving cells is regulated by selecting the lightdose in relation to the concentration of the photosensitivity agent.Again, such techniques are known in the art.

The present invention provides an efficient means for delivery of alarge variety of antigenic molecules. The invention has a number offeatures rendering it particularly suitable as a vaccine deliverytool: 1) it has no restrictions on the size of the molecule to bedelivered as long as the molecule can be endocytosed by the target cell;2) it is not dependent on cell proliferation; 3) it is site specific inthat only areas exposed to light are affected; 4) it is not oncogenic.In addition, photochemical internalisation may potentially be combinedwith other principles for generating site or tissue specific drugaction, such as targeting by the use of specific ligands for cellsurface structures, employing regulatory gene elements that confertissue specificity or the use of disease-specific drugs, opening apossibility of obtaining substantially synergistic effects in thespecificity of drugs for target cells.

The invention will now be described in more detail in the followingnon-limiting Examples with reference to the following drawings in which:

FIG. 1 shows a schematic representation of how PCI can be utilised tostimulate CTLs. A peptide or protein (P) is applied extracellularly toantigen presenting cells. P is endocytosed and released, into cytosol byPCI. The peptide or protein will thereafter be partly degraded byproteasomes and transported to the cells surface complexed to MHC (HLA)class I where, the complex can be recognised by CTLs.

FIG. 2 shows photochemically induced relocalization of a peptide.BL2-G-E6 cells were incubated with a fluorescein-labelledp21^(ras)-derived 5-21, Val¹² peptide and AlPcS_(2a). The cells wereexamined for fluorescein-peptide and AlPcS_(2a) localisation byfluorescence microscopy before (top panels) and 30 minutes after (bottompanels) a 4-min exposure to red light. Bar 20 μm.

FIG. 3 shows the cytotoxicity of a CD8⁺ T lymphocyte clone against FM3melanoma cells after PCI of a MART-1 peptide.

FIG. 4 shows the ability of PCI to deliver HRP into the cytosol. NHIK3025 cells were treated with 3.2 μg/ml TPPB_(2a) and 1 mg/ml HRP for 18hours. The medium was then replaced with drug-free medium beforeexposure to the indicated light doses. HRP activity was measured inintact cells (∘) and in cytosol () separated from cytosol-free cellcorpses (▾) by electropermeabilisation and a density centrifugationtechnique.

FIG. 5 shows photochemically induced expression of GFP. a. expression ofGFP in THX cells treated with pEGFP-N1-pLys complex in the absence ofAlPcS_(2a) and light or in the presence of AlPcS_(2a) followed byexposure to light as indicated on the figure. The cells were analysed byflow cytometry, reckoning the cells on the right side of the drawn lineas positive for GFP expression. b. expression of GFP in THX cellstreated for 18 hours with a photosensitizer (20 μg/ml AlPcS_(2a) or 0.25μg/ml 3-THPP) followed by a 6 hour transfection with pEGF-N1-pLyscomplex and exposure to light inactivating 50% of the cells. GFPexpression was analysed by flow cytometry as described in a.

EXAMPLES Materials and Methods Irradiation

Two different light sources were used for treatment of the cells, bothconsisting of a bank of 4 fluorescent tubes. Cells treated with TPPS₄,TPPS_(2a), and 3-THPP (Porphyrin Products, Logan, Utah) were exposed toblue light (model 3026; Appl. Photophysics, London, UK) with a lightintensity reaching the cells of 1.5 mW/cm² while cells treated withAlPcS_(2a) (Porphyrin Products, Logan, Utah) were exposed to red light(Philips TL 20W/09) filtered through a Cinemoid 35 filter with a lightintensity reaching the cells of 1.35 mW/cm².

Fluorescence Microscopy

The cells were analysed by fluorescence microscopy as described in Berg.K., et al., Biochem. Biophys. Acta., 1370: 317-324, 1998. For analysisof fluorescein-labelled molecules the microscope was equipped with a450-490 nm excitation filter, a 510 nm dichroic beam splitter and a510-540 nm band pass emission filter.

Preparation of Plasmid-pLys Complexes and Treatment of Cells

Plasmid-pLys complexes (charge ratio, 1.7) were prepared by gentlymixing 5 μg plasmid (pEGFP-N1; Clontech Laboratories, Inc., Palo Alto,Calif.) in 75 μl of HBS with 5.3 μg pLys (mW 20700; Sigma, St. Louis,Mo.) in 75 μl of HBS. The solutions were incubated for 30 min at roomtemperature, diluted with culture medium and added to the cells.

THX cells were incubated with 20 μg/ml AlPcS_(2a) for 18 hours at 37°C., washed and incubated in sensitizer-free medium for 3 hours beforeincubation with plasmid-pLys complexes for 2 hours. The pEGFP-N1/pLystreated THX cells were washed once and incubated for 2 hours in culturemedium without additions before exposure to light. The cells wereincubated at 37° C. for 2 days, subcultured and further incubated for anadditional 5 days before analysis of GFP expression by flow cytometry.

HCT-116 cells were incubated with 20 μg/ml AlPcS_(2a) for 18 hours,washed and transfected with plasmid-pLys complexes for 6 hours beforelight exposure in plasmid-free medium. After 40 hours incubation at 37°C. the GFP expression was studied by microscopy.

Flow Cytometry Analysis

The cells were trypsinized, centrifuged, resuspended in 400 μl ofculture medium and filtered through a 50 μm mesh nylon filter. The cellswere then analysed in a FACStar plus flow cytometer (Becton Dickinson).Green Fluorescent Protein (GFP) was measured through a 510-530 nm filterafter excitation with an argon laser (200 mW) tuned on 488 nm.AlPcS_(2a) was measured through a 650 nm longpass filter afterexcitation with a krypton laser (50 mW) tuned on 351-356 nm. Celldoublets were discriminated from single cells by gating on the pulsewidth of the GFP fluorescence signal. The data were analysed with PCLysys II software (Becton Dickinson).

Preparation of Fluorescein-Peptide and Treatment of Cells

The fluorescein-labelled Val¹²-p21^(ras)-peptide (residues 5-21) weresynthesised and provided by Alan Cuthbertson, Nycomed Amersham).

BL2-G-E6 cells were incubated with 30 μg/ml of the fluorescein-labelledp21^(ras)-derived peptide for 18 hours followed by 20 μg/ml AlPcS_(2a)for 18 hours and 1 hour in drug-free medium before exposure to redlight.

Example 1 Photochemical Internalisation (PCI) can be Used to EnablePeptides to Enter the Cytosol of Cells

To evaluate PCI for cytosolic delivery of cancer-specific peptides, afluorescein-labelled p21^(ras) peptide encompassing residues 5-21 andcontaining a Val¹² mutation (G12V) was used (Gjertsen, M. K., et al.,Int. J. Cancer, 72: 784-790, 1997). In BL2-6-E6 mouse fibroblasts, theras peptide colocalised well with AlPcS_(2a), indicating endocyticuptake of the peptide (FIG. 2). After a 4-min exposure to light, thefluorescein-labelled ras peptide and AlPcS_(2a) were found to be locateddiffusely in the cytoplasm. Similar effects were not Observed in cellsexposed to the fluorescein-labelled ram peptide and light only (data notshown).

Example 2 Use of PCI to Induce Antigen Presentation and CD8⁺ TLymphocyte Mediated Cell Killing

FM3 melanoma cells (2×10⁵/well in 6 well plates), grown in RPMI 1640medium with 10% foetal calf serum (FCS), not expressing MART-1 peptidewere treated with 10 μg/ml of the photosensitizing agent AlPcS_(2a) for18 hours. The cells were then released from the substratum with EDTA(0.1 M) in Dulbecco's phosphate-buffered saline (PBS) and kept insolution during loading of the cells with ⁵¹Cr (60 μCi/ml Na₂CrO₄) for 1hour in 100% FCS followed by 5 hours incubation with 5 μg/ml MART-1peptide in RPMI 1646 in 10% FCS, while the cells were still kept insolution. The sequence of the MART-1 peptide was: TAEEAAGIGILTVILG. Thecells were then washed twice in RPMI 1640 medium containing 10% FCS andseeded out in 96-well plates (2000/well in 100 μl medium (RPMI 1640/10%FCS). The cells were then exposed to light for the times as indicated inFIG. 3 ((Philips TL 20W/09) filtered through a Cinemoid 35 filter with alight intensity reaching the cells of 1.35 mW/cm² (Rodal et al., 1998,J. Photochem. Photobiol. B: Biol. 45: 150-9)). 18 hours after lightexposure the medium was removed and medium containing MART-1/HLA-A2specific cytotoxic T lymphocytes (CTLs—40,000/well added in 100 μl) wereadded. After 4 hours of incubation the medium was separated from FM3cells and the ⁵¹Cr released to the medium (as an indicator of lysedcells) was counted as well as the spontaneous and maximum release aspreviously described (Possum et al., 1995, Cancer Immunol. Immunother.40: 165-172). The percentage specific chromium release was calculated bythe formula: (experimental release−spontaneous release)/(maximumrelease−spontaneous release)×100. It can be seen from the results shownin FIG. 3 that FM3 cells after PCI of a MART-1 peptide as outlined aboveshow light dependent susceptibility to CD8⁺ T lymphocyte cytotoxicity.

Example 3 PCI Induces the Release of a Large Fraction of the EndocytosedMolecule

This was shown by PCI induced internalisation/endocytosis of HorseradishPeroxidase (HRP).

By using HRP, it is demonstrated (see FIG. 4) that PCI induces therelease of a large fraction (>60%) of endocytosed HRP into the cytosol.

In this experiment NHIK 3025 cells (carcinoma cells in situ from humancervix) were treated with the photosensitizing agent TPPS₂, (3.2 μg/ml)and 1 mg/ml HRP for 18 hours. The medium was then replaced with drugfree medium before exposure to the light doses as indicated in FIG. 4,HRP activity was measured according to the procedure described inSteinman et al., J. Cell. Biol., 68: 665-687, 1976. Cytosol wasseparated from cytosol-free cell corpses by electropermeabilisation anda density centrifugation technique (Berg et al., Int. J. Cancer 59:814-822, 1994).

Example 4 PCI can be Used to Enhance the Delivery of Functional Genes

To demonstrate this, THX cells were transfected with a pLys-complex of aplasmid (pEGFP-N1) coding for green fluorescent protein (GFP). Theexpression of GFP was analysed by flow cytometry (FIG. 5, a and b) andfluorescence microscopy (data not shown). As can be seen from FIG. 5 a,treatment with, AlPcS_(2a) and light led to a strong increase in thepercentage of the cells expressing GFP. The fraction of the cells thatwas positive for this reporter molecule increased from 1% at no lighttreatment to 50% after a 5-min light exposure. GFP expression was notenhanced by light in cells treated with pEGFP-pLys in the absence of aphotosensitizer. A complex of an irrelevant plasmid (encoding hemsoxygenase) and pLys did not induce green fluorescence when combined withAlPcS_(2a) and light (data not shown). Consequently, in a light-directedmanner, PCI can substantially increase the efficiency of transfection ofa functional gene to THX cells. Similar results were obtained usingTPPS_(2a) as a photosensitizer and BHK-21 and HCT-116 as target cells(data not shown). The essentially non-lysosomally located sensitizer3-THPP induced only a minor increase in GFP expression (FIG. 5 b). PCIof pEGFP-N1 not complexed with pLys did not induce the expression of GFP(data not shown).

1.-52. (canceled)
 53. A method of stimulating an immune response to anantigenic molecule in vivo, said method comprising: contacting a cellwith said antigenic molecule and with a photosensitizing agent ex vivo,wherein said molecule and said agent are each taken up into anintracellular membrane-restricted compartment of said cell; irradiatingsaid cell ex vivo with light of a wavelength effective to activate thephotosensitizing agent, such that the membrane of said intracellularcompartment is disrupted, releasing said molecule into the cytosol ofthe cell, without killing the cell; wherein said released antigenicmolecule, or a part thereof of sufficient size to stimulate an immuneresponse, is subsequently presented on the surface of said cell;administering the cell to a mammal after irradiating said cell tothereby stimulate the in vivo immune response to the antigenic molecule.54. The method of claim 53, wherein the antigenic molecule is apolypeptide.
 55. The method of claim 53, wherein the antigenic moleculeis a vaccine antigen or vaccine component.
 56. The method of claim 53wherein the cell is an antigen presenting cell selected from the groupcomprising lymphocytes, dendritic cells, macrophages and cancer cells.57. The method of claim 53 wherein the photosensitizing agent isselected from the group consisting of porphyrins, phthalocyanines,purpurins, chlorins, benzoporphyrins, naphthalocyanines, cationic dyes,tetracyclines and lysomotropic weak bases or derivatives thereof. 58.The method of claim 53 wherein the photosensitizing agent ismeso-tetraphenylporphine with 4 sulfonate groups (TPPS₄),meso-tetraphenylporphine with 2 sulfonate groups on adjacent phenylrings (TPPS_(2a)), or aluminum phthalocyanine with 2 sulfonate groups onadjacent phenyl rings (AlPcS_(2a)).
 59. The method of claim 53, whereinthe antigenic molecule and/or photosensitizing agent is bound to one ormore targeting agents or carrier molecules.
 60. The method of claim 53,wherein at least 90% of the cells are not killed.
 61. The method ofclaim 53, wherein at least 95% of the cells are not killed.
 62. Themethod of claim 53, wherein the photosensitizing agent is a sulfonatedtetraphenylporphine, a disulfonated aluminum phthalocyanine or atetrasulfonated aluminum phthalocyanine.
 63. The method of claim 53,wherein the antigenic molecule stimulates cytotoxic T cells.
 64. Amethod of stimulating an immune response to an antigenic molecule, saidmethod comprising: contacting a cell with an antigenic molecule and witha photosensitizing agent, wherein said antigenic molecule and said agentare each taken up into an intracellular membrane-restricted compartmentof said cell; irradiating said cell with light of a wavelength effectiveto activate the photosensitizing agent, such that the membrane of saidintracellular compartment is disrupted, releasing said antigenicmolecule into the cytosol of the cell, without killing the cell; whereinsaid released antigenic molecule, or a part thereof of sufficient sizeto stimulate an immune response, is subsequently presented on thesurface of said cell; wherein presentation of the antigenic molecule, orpart thereof, on the surface of said cell results in stimulation of theimmune response specific for said antigenic molecule or a part thereof.65. The method of claim 64, wherein the antigenic molecule is apolypeptide.
 66. The method of claim 64, wherein the antigenic moleculeis a vaccine antigen or vaccine component.
 67. The method of claim 64wherein the cell is an antigen presenting cell selected from the groupcomprising lymphocytes, dendritic cells, macrophages and cancer cells.68. The method of claim 64 wherein the photosensitizing agent isselected from the group consisting of porphyrins, phthalocyanines,purpurins, chlorins, benzoporphyrins, naphthalocyanines, cationic dyes,tetracyclines and lysomotropic weak bases or derivatives thereof. 69.The method of claim 64, wherein the photosensitizing agent ismeso-tetraphenylporphine with 4 sulfonate groups (TPPS₄),meso-tetraphenylporphine with 2 sulfonate groups on adjacent phenylrings (TPPS_(2a)), or aluminum phthalocyanine with 2 sulfonate groups onadjacent phenyl rings (AlPcS_(2a)).
 70. The method of claim 64, whereinthe antigenic molecule and/or photosensitizing agent is bound to one ormore targeting agents or carrier molecules.
 71. The method of claim 64,wherein at least 90% of the cells are not killed.
 72. The method ofclaim 64, wherein at least 95% of the cells are not killed.
 73. Themethod of claim 64, wherein the photosensitizing agent is a sulfonatedtetraphenylporphine, a disulfonated aluminum phthalocyanine or atetrasulfonated aluminum phthalocyanine.
 74. The method of claim 64,wherein the antigenic molecule stimulates cytotoxic T cells.
 75. Themethod of claim 64 wherein said method is carried out in vitro or invivo.
 76. A method of producing a cell expressing an antigenic moleculeor a part thereof on the surface of said cell, said method comprising:contacting a cell with an antigenic molecule and with a photosensitizingagent, wherein said antigenic molecule and said agent are each taken upinto an intracellular membrane-restricted compartment of said cell;irradiating said cell with light of a wavelength effective to activatethe photosensitizing agent, such that the membrane of said intracellularcompartment is disrupted, releasing said antigenic molecule into thecytosol of the cell, without killing the cell; wherein said releasedantigenic molecule, or a part thereof of sufficient size to stimulate animmune response, is subsequently presented on the surface of said cell;wherein the cell is an antigen presenting cell selected from the groupconsisting of lymphocytes, dendritic cells and macrophages.
 77. A kitfor use in expressing an antigenic molecule or a part thereof on thesurface of a cell, said kit comprising: a first container containingsaid antigenic molecule; and a second container containing aphotosensitizing agent.
 78. A composition for expressing an antigenicmolecule or a part thereof on the surface of a cell, said compositioncomprising an antigenic molecule and a photosensitizing agent.